Production of edible protein from non-toxic strains of penicillium

ABSTRACT

Penicillin free edible protein-containing substance suitable for human consumption is produced by incubating and proliferating, under aerobic conditions, a non-toxic and non-penicillin producing strain of Penicillium notatum or Penicillium chrysogenum or a variant or mutant thereof in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which is penicillin free and which constitutes the edible protein-containing substance suitable for human consumption possessing a high net protein utilization value of at least 70 based on alpha-amino nitrogen.

United States Patent 1 1 Y Spicer et al.

[63] Continuation of Ser. No. 116,684, Feb. 18, i971,

abandoned.

' V [30] Foreign Application Priority Data Feb. 25, 1970 Great Britain 8977/70 Feb. 25, 1970 Great Britain 8978/70 s2 11.s.'c1... 426/60, 195/35, 426/204 51 Int. Cl. C.12j 13/06,A23j 3/00 [58] Field of Setll'cli.... 99/14; 195/27, 35, 81;

[56] i 9 References Cited UNITED STATES PATENTS 3,151,038

9/1964 Gray 195/81 X I 451 Febrli", 197s 0THER PUBLICATIONS Fink et al., Chemical Abstracts, Vol. 48, No. 11593C, 1954. Y I

Primary Examiner-James R. Hoffman I Attorney, Agent, or Firm-Stevens, Davis,--Miller' & Mosher v [57] ABSTRACT -v I v Penicillin free edible protein-containing substance suitable for human consumption is produced by incubating and proliferating, under aerobic :conditions, a non-toxic and non-penicillin producing strain of Penicillium notarum or Penicillium chrysogenum or a variant or mutant thereof in a culture medium containing essential growth-promoting nutrient substances, of

which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which is penicillin 'free and which constitutes the edible protein-containing substance suitable forhuman consumption possessing a high net protein utilization value of at least 70 based on alpha-amino nitrogen.

"5 Claims, No Drawings protein by microbial action.

Our British Application No. 53312/66, now- British Pat No. 1210356, relates to a process for the production of an edible protein-containing substance which comprises incubating and proliferating, under aerobic conditions, an organism which is a non-toxic strain of a microfungus of the class Fungi Imperfecti, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from a assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible proteincontaining substance.

It is an object of the present invention to provide fungal mycelium as an unsupplemented protein source which possesses a high net protein utilisation value on rat assays of at least 70 based on the a-amino nitrogen.

According to the present invention there is provided a process for the production of an edible proteincontaining substance which comprises incubating and proliferating, under acrobic conditions, a non-toxic strain of Penicillium' notatum or Penicillium chrysogenum or a variant or mutant thereof, in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which constitutes the edible protein-containing substance.

The separated proliferated organism which constitutes the edible protein-containing substance may be incorporated into a foodstuff for human or animal consumption.

The substrate employed in the incubation stage may be of vegetable origin for example wheat feed, hydrolysed potato, molasses, bagasse waste and/or citrous waste. Alternatively a substrate of animal origin comrising whey may be employed.

Our strain of Penicillium notatumchrysogenum C1 which is described together with variants and mutants thereof in British Application No. 8978/70, now British Pat. No. 1,331,472, has been deposited at at Commonwealth Mycological Institute, and assigned the number I.M.I. 138291.

It has the following morphological characteristics:

Close textured, velvety Radial furrows in Close textured. velvety No radial furrows Character of growth -Continued Medium Malt Extract Czapek-Dox (modified) Agar (Oxoid) Agar-(Oxoid) some cases v Margin Entire. 2-3 m. Entire. l-2mm white,

white, non-sporing non-sparing except except where where colonies meet colonies meet I Amount of sporulati Dense Dense Color Blue-green to Blue-green bright green Exudate Absent Present, clear yellow,

mainly in separated colonies Odor Very little Very little Reverse Yellow to yellow- Bright yellow with brown, no pigment abundant pigment visible in medium diffusing into medium Conidial Stage Very similar on both media Penicilli: Biverticillate asymmetrical. Length (branch to phialides) 16-35p.

Conidiophore: At colony margin arising from basal felt, smooth-walled, 200-3001.. approx. Width Branches: One or two branches usually, arising at same point on main stem, asymmetric. Length 920p., width 2-4 Melulae: Groups of 2-5, usually 3. Compact whorl, not divergent, length 3-10u, width 2-4;

Pltialide.r: Groups of 4-6, typical bottle-shaped cells. Lengths 4.5-6.0p, width 2.0-3.0;1.

Conidia: Forming tangled chains up to 120p. in

length. usually 80p, Subglobose, 3-3.5 X-2.5-3p., g smooth walled, yellowish green in mass The penicillr' are intermediate between P. Chrysogenum and notatum in complexity with slightly elliptical spores lilre clrrysogenum numbers I.M.l. 142383 and 142384 respectively. and 1.

Sporulation Medium Malt Extract Czapek-Dox (modified) Agar (Oxoid) Agar (Oxoid) Growth conditions 10 days at 25C 10 days at 25C Colony characteristics Rate of growth 5.0-6.0 cm 4.5-50 cm in 10 days in 10 days Character of Floccose at first, Floccose particularly growth tending to become in center. Four radial velvety. Light furrows. radial furrows Margin Entire v Entire Amount of Sparse Sparse -Continued -Continued 1. 113 l. 113 Medium Malt Extract Czapek-Dox (modified) Medium Malt Extract Czapek-Dox (modified) Agar (Oxoid) Agar (Oxoid) 5 Agar (Oxoid) Agar (Oxoid) Color White to pale White to pale lemon Conidia: Very short clti ains,s one to threse spores, 5.s T8',l l'.wJri will??? Exudate Absent 'Absent very very The mutant l. 64 (I.M.I. 142,383) has a morphology Reverse Y -b Bright y w with, identical to its parent CI. The mutant I. 156 has a morp'gmem m medlum 22332: p'gmem m phology very similar to I. 1 13 except the colony is yellow-brown to cream in color in contrast to the pale Qwe Sirnilar on both media green color of I 1.31 Penifl-m: Bivemcmm asymmetric. Length 15 The temperature of incubation is in general between (branch to phialide) -50;. and 35C., preferably around C. conidiophme: smooth waned, approx 200 width 24 Inoculation resulting in commencement of the process is best carried out by a pregermmated seedstage BranchesI 3 2) g f z zgi fi gg g' gi 225 20 comprising for example from 2 percent to 10 percent 2030 gccasionauy ve f gt of inoculum, usually in the range 5 percent to 10 per- Width 3-4 cent. Metal: Usually we occasionally three pet The pH of the substrate medium during incubation is branch. Len th 5-l0p. usually 10 preferably kept within a suitable range supporting max- Width 4 as some are swollenln cent" imum growth, for example, between 4 to 7. Phialides: One or two per metula, typical bottle 25 The period of growth in batch culture is usually shape. Length 4-8;. Width 2-3; found to range from 20 to 48 hours. ln certain instances Canidia: Short chains up to 60M length for example when the carbohydrate is in the form of usually 25-30;. Subglobose 2.5-3.5;1. lactose-the time may be extended to 72 hours. In both X Smooth Walled batch and continuous processes aeration and agitation 1 '95 30 should be carried out to provide a sufficient level of dis- M Ma" Extract Czapek Dex difi d) solved oxygen to overcome oxygen deficiency which is g g a limiting growth factor. Growth As will be well understood by those skilled in the art conditions 10 da s at 25C 10 days at 25C sufficient quantltles of essentlal growth nutrients such Colony as mtrogen, sulphur, phosphorus and other trace elecharacteristics ments are maintained in the substrate medium so that m cm 5 cm in 10 days growth of the substance is limited only by the carbohyin 10 y drate available to the fungus. Character of In addition to the nutrients stated above. the presgrowth Floccose center As on M.E.A. ence of one or more vitamins such for example as bioz fi 'i 2 Many radial furrows tin may be desirable to maintain maximum growth rate. radial furrows It is also desirable to add a non-toxic antifoaming agent to the substrate medium to control foaming dur- Margln Entlre Crenate g the fermentation. A twu 0f po y sp se. As on MBA The substance produced according to the present in- 31mm Sterne vention may be isolated in any suitable manner known Color White White center, in the art. Thus the resultant mycelium may be recovmargin ered by separation, washing, filtration and drying. In this connection, however, it has been found that if the Exudate Absent Absent moisture content of the substance is reduced below a Odor verymfle verymfle critical level of about 50 percent (w/w) by filtration under pressure the subsequent drying methods em- Revme g'g g gf m fi ployed are not subjected to such stringent temperature produced limitations which is an important factor in the ecoconidialmge nornlc processing of these materials. The method of W Assymmemcal Lfingth (branch to drylng must not cause damage to the nutritional value phialides) 20-50 of the mycelium and may be drying 1n a current of air Conidiophore: Smooth walled approx. 2 300p. arising from at 75C or freeze d i hyphae' w'dth The fungal mycelium produced by the process of the Branches: Single arising from several points on present invention shows very good water binding caf gg zfgi ig a fi gl i pacity and may be useful as a thickening and gelling agent. Not being an isolate, it retains its vitamins as well Me'ulaef one f a fi z lg% 34 as other nutritionally available materials such as lipids 63:5 ;,1;, I 65 and some carbohydrates. Fungal mycelium has satisfactory baking characteristics which are of value in pro- Phialides: One per metula, rather restricted in comparison with typical shape. Length 4-8;; Width 2p.

tein enriched breads, breakfast foods and food snacks. The fungal mycelium, because of its filamentous struc- EXAMPLE 1 Litres of the following culture medium were prepared and sterilised in a stirred fermenter vessel.

Cane molasses to provide 6% w/v sugar Ammonium sulphate 1.2 Potassium dihydrogen 0.3%

phosphate Calcium carbonate 1.0% (to control pH.

otherwise gaseous ammonia could be used on automatic control) Tap water Antifoam: Polypropylene glycol 2000 2 ml. was

added to prevent foam formation An inoculum equivalent to 5-l0 percent by volume of the culture medium and grown either on a glucose/- corn steep liquor salts medium or other suitable nutrients in shake flasks was inoculated with a spore suspension of the organism, comprising our strain of Penicil- Iium notatum-chrysogenum, Cl, for 18 to 24 hours at 30C. on a rotary shaker.

The fermenter incubated at 30C. was then stirred at 800 rpm with a disc turbine and l VVM of sterile air passed through. in 30 to 40 hours, the grown mycelium was removed from the fermenter, centrifuged, washed with water and dried in a current of air at 75C.

EXAMPLE 2 This is an example of a starch preparation using potatoes as the starch source.

Washed potatoes were placed with water in a jacketed stirred vessel, brought to boiling with stirring and boiled until the whole was a uniform mash. The temperature was then reduced to 65C., pH adjusted to 6.0 6.5 and heat stable bacterial a-amylase added and the stirring continued. After 20 minutes the liquified material was passed through a sieve in order to remove the potato skins, which are not attacked by the enzyme. This solubilised solution was then adjusted with water to give a solution that contained 6 percent of sugars.

400 1. of the'following culture medium were prepared and sterilised in a stirred fermenter vesselzPotato (treated as described above to provide) 60% w/v. sugar Ammonium sulphate 0.25%

KHJO. 0.3% Tap water Antifoam soyabean oil (sufficient tov prevent excessive foaming) Analysis Total Nitrogen (Kjeldahl) 6.90% 0: Amino Nitrogen 5.27%

Sulphur amino acids represent 3.40 percent of the total amino acids.

EXAMPLE 3 400 1. of the following culture medium were preizersfl ns sfi z in a S ed r er Cane molasses to provide 6.0% w/v sucrose 2000 (sufficient to prevent excessive foaming) Analysis Total Nitrogen (Kjeldahl) a Amino Nitrogen Sulphur amino acids represent 2.37 percent of the total amino acids.

EXAMPLE 4 400 l. of the following culture medium were pre- Spray dried milk whey (7% w/v) (to provide) Corn steep liquor (50% T5) 6.0% w/v lactose 0.4%

Ammonium sulphate 0.4%

Tap water Antifoam Polypropylene glycol 2000 (sufficient to prevent excessive foaming) pH 5.5 (no control required). The process and other conditions were as described in Example 2. At approximately 72 hours mycelium was recovered as in Example 2.

Analysis Total Nitrogen (Kjeldahl) a Amino nitrogen Sulphur amino acids represent 4.31 percent of the total amino acid EXAMPLE 5 8 l. of the following culture medium were prepared and sterilized and added to a sterile fermenter.

(su icient to prevent excessive foaming) pH maintained at 5.0.with gaseous ammonia. The seed inoculum was percent of our strain of Penicillium notatum chrysogenum 1.1 13 (lMl 1423 85). The fermenter temperature was controlled at 30C. The fermenter was stirred at speeds of up to 800 rpm. with slow increase of the stirrer speed during the course of the batch to maintain the level of dissolved oxygen in the culture at a non-growth limiting concentration. The impeller was a single disc-turbine impeller of 1 1.5 cm. diameter. Aeration was at 1 VVM. After 25.5 hours a sample of the mycelium gave, on a dry weight basis a total Nitrogen of 7.51 percent.

EXAMPLE 6 The procedure of Example 5 was repeated with the exception that the culture used was our strain of Penicillium notatum chrysogenum 1.195 (IMI 142386) and the ph was controlled at 6.0. After 20.5 hours a sample of the mycelium gave, on a dry weight basis, a total Nitrogen of 7.53 percent. 7

Our strain of Penicillium notatum chrysogenum C 1 and the variants thereof may be replaced in the above examples by other strains of Penicillium notatum and Penicillium chrysogenum and Penicillium notatum chrysogenum that are non-toxic and contain no other undesirable metabolites such for example as the antibiotic penicillin.

What we claim is:

l. A process for the production of a penicillin free for human consumption which comprises incubating and proliferating, under aerobic conditions, a non-toxic and non-penicillin producing strain of Penicillium notatum or Penicillium chrysogenum or a variant or mutant thereof in a culture medium containing essential growth-promoting nutrient substances, of which carbon in the form of assimilable carbohydrate constitutes the limiting substrate in proliferation, and separating from the assimilable carbohydrate exhausted medium the proliferated organism which is penicillin free and which constitutes the edible protein-containing substance suitable for human consumption possessing a high net protein utilization value of at least based on a-amino nitrogen.

2. A process as claimed in claim 1 wherein the separated proliferated organism which constitutes the edible protein-containing substance suitable for human consumption is incorporated into a foodstuff for human or animal consumption.

3. A process as claimed in claim 1 wherein the nontoxic strain is the strain of Penicillium notatumchrysogenumCl deposited with the Commonwealth Mycological Institute and assigned the number l.M.l. 138291.

4. A process as claimed in claim 1 wherein the nontoxic strain is a variant [.113 or 1.195 of the strain of Penicillium notatum-chrysogenum C l deposited with the Commonwealth Mycological Institute and assigned the numbersLMl. 142385 and I.M.I. 142386 reps ve w 5. A process as toxic strain is a mutant 1.64 or 1. 156 of the strain of Penicillium notatum-chrysogenum Cl deposited with the Commonwealth Mycological Institute and assigned the numbers I.M.I. 142383 and I.M.I. 142384 respectively.

claimed in claim 1 wherein the non- 

1. A PROCESS FOR THE PRODUCTION OFF A PENICILLIN FREE EDIBLE PROTEIN-CONTAINING SUBSTANCE WHICH IS SUITABLE FOR HUMAN CONSUMPTION WHICH COMPRISES INCUBATING AND PROLIFERATING, UNDER AEROBIC CONDITIONS, A NON-TOXIC AND NON-PENICILLIN PRODUCING STRAIN OF PENICILLIN NOTATUM OR PENICILLIUM CHRYSOGENUM OR A VARIANT OR MUTANT THEREOF IN CULTURE MEDIUM CONTAINING ESSENTIAL GROWTH-PROMOTING NUTRIENT SUBSTANCES OF WHICH CARBON IN THE FORM OF ASSIMILABLE CARBOHYDRATE CONSTITUTES THE LIMITING SUBSTRATE IN PROLIFERATION, AND SEPARATING FROM THE ASSIMILABLE CARBOHYDRATE EXHAUST MEDIUM THE PROLIFERATED ORGANISM WHICH IS PENICILLIN FREE AND WHICH CONSTITUTES THE EDIBLE PROTEIN-CONTAINING SUBSTANCE SUITABLE FOR HUMAN CONSUMPTION POSSESSING A HIGH NET PROTEIN ULTILIZATION VALUE OF AT LEAST 70 BASED ON A-AMINO NITROGEN.
 2. A process as claimed in claim 1 wherein the separated proliferated organism which constitutes the edible protein-containing substance suitable for human consumption is incorporated into a foodstuff for human or animal consumption.
 3. A process as claimed in claim 1 wherein the non-toxic strain is the strain of Penicillium notatum-chrysogenumC1 deposited with the Commonwealth Mycological Institute and assigned the number I.M.I.
 138291. 4. A process as claimed in claim 1 wherein the non-toxic strain is a variant I.113 or I.195 of the strain of Penicillium notatum-chrysogenum C1 deposited with the Commonwealth Mycological Institute and assigned the numbers I.M.I. 142385 and I.M.I. 142386respectively.
 5. A process as claimed in claim 1 wherein the non-toxic strain is a mutant I.64 or I. 156 of the strain of Penicillium notatum-chrysogenum C1 deposited with the Commonwealth Mycological Institute and assigned the numbers I.M.I. 142383 and I.M.I. 142384 respectively. 